Publications [#232645] of Michael C. Fitzgerald

Journal Articles

  1. Stamps, SL; Fitzgerald, MC; Whitman, CP, "Characterization of the role of the amino-terminal proline in the enzymatic activity catalyzed by macrophage migration inhibitory factor.", BiochemistryJuly,, 1998, 37(28), 10195-10202 [doi].
    (last updated on 2024/04/22)

    Abstract:
    The cytokine macrophage migration inhibitory factor (MIF) mediates several immune and inflammatory processes through unknown or poorly understood mechanisms. The protein shares structural homology with two bacterial isomerases, 4-oxalocrotonate tautomerase (4-OT) and 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and catalyzes the enolization of phenylpyruvate and the ketonization of (p-hydroxyphenyl)pyruvate. The amino-terminal proline has been identified as the catalytic base in both the 4-OT- and CHMI-catalyzed reactions. MIF also has an amino-terminal proline that has been implicated as a catalytic group in the MIF-catalyzed reaction. To delineate further the role of Pro-1 in the MIF-catalyzed reaction, affinity labeling studies were performed with 3-bromopyruvate (3-BP). The results of this study show that 3-BP acts as an active-site-directed irreversible inhibitor of the enzymatic activity and modifies one site per monomeric subunit. The inhibitor, as its lactyl derivative, is covalently attached to an 11 residue amino-terminal fragment, Pro-1 to Arg-11. The only reasonable site for alkylation within this peptide fragment is the amino-terminal proline. Because the pKa measured for the pH dependence of kinact/KI (5.7 +/- 0.2) and that measured for the pH dependence of the kcat/Km for the enolization of phenylpyruvate (6.0 +/- 0.1) are comparable and in reasonable agreement with the previously measured pKa of Pro-1 (5.6 +/- 0.1) obtained by its direct titration [Swope, M., Sun H.-W., Blake, P., and Lolis, E. (1998) EMBO J. (in press)], it is concluded that Pro-1 acts as the general base catalyst in the MIF-catalyzed reaction. The structural and mechanistic parallels place 4-OT, CHMI, and MIF in a superfamily of enzymes related by their ability to catalyze the keto-enol tautomerization of a pyruvyl moiety.